The interaction of trans-ddp [PtCl2(NH3)2] with low doses of radiation in mammalian cells

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In/. J Radralion Oncology Biol. Phys., Vol. 16, PP. 1281-1284 Printed in the U.S.A. All rights reserved.

??Session VI THE INTERACTION OF TRANS-DDP [PtC12(NH3)2] WITH LOW DOSES OF RADIATION IN MAMMALIAN CELLS K. A. SKOV, PH.D.,* B.C. Cancer

Research

M.

Centre,

KORBELIK,

PH.D.?

601 West 10th Avenue,

AND B. PALCIC,

Vancouver,

PH.D.-+

B.C. V5Z lL3 Canada

Trans-DDP, a less toxic isomer of cisplatin, was examined for its radiosensitizing activity at high and low doses of ionizing radiation. Cells were exposed to the drug to produce low toxicity before exposure to X rays. A sensitive assay using the DMIPS Cell Analyzer was employed to measure cell survival response in low dose region (O-4 Gy) and conventional assay was used for high doses (4-25 Gy). The results show that trans-DDP is a much more effective radiosensitizer at low doses than at high doses, whether or not cytotoxicity was pronounced. This is in contrast to any other sensitizer studied to date, including oxygen, misonidazole, SR-2508 and Ro-03-8799, regardless of prior incubation and/or cytotoxicity. Platinum complexes, Ionizing radiation, Low doses, Shoulder region, Radiosensitization, INTRODUCTION

Cytotoxicity.

For example, the oxygen enhancement ratio (OER) is reduced from approximately 3.5 to 2.4 at survival levels of 0.0 1 to 0.8, respectively (15). Similarly, misonidazole was shown to be much less effective at low doses; if all survival curves were analyzed by the linear quadratic equation, the (Yparameter was not affected by the presence of misonidazole up to 5 mM concentration; the whole radiosensitizing effect could be attributed to the modification of the p parameter (12). However, if incubation of cells at 37°C with misonidazole was permitted prior to irradiation the CYparameter was also affected (Table 1, footnote vi). Similar observations were made for nitroimidazoles SR-2508 and Ro-03-8799 (18). In each case, however, using nitroimidazoles, radiosensitization at high doses was greater than that at low doses. Fractionated studies using cis-DDP in vivo suggested even better enhancement ratio than single exposure (3); thus this drug may be a better sensitizer at low doses than at high doses. To check this hypothesis, we first used the less toxic trans-DDP, a geometric isomer of the clinically active cis-DDP, and performed studies at low and high radiation doses. Trans-DDP exhibits radiosensitizing activity (4), as do some second generation clinically active compounds (6), and may act with radiation similar to the cis-DDP.

The potential of cis-diamminedichloroplatinum(II), cisDDP, as a chemotherapeutic agent was recognized in 1965 by Rosenberg ( 17). The drug was introduced into the clinic as cisplatin before 1972 (9). The first report on the interaction of this compound with ionizing radiation was in 197 1 (20). Since then, it has been reported that this interaction also occurs in humans. A good summary of these interactions can be found in recent reviews (3, 1 l), describing in vitro, in vivo and clinical studies to date. In general, radiosensitization in vitro has been studied using a clonogenic assay. This assay, as practiced in a conventional way, gives very good experimental data at large radiation doses, where most of the cells are inactivated. At therapeutically relevant dose levels (l-3 Gy), where most cells survive, this assay gives inaccurate data due to large experimental errors and statistical uncertainties (13). To circumvent this problem, we used the DMIPS Cell Analyzer which was specifically designed to detect and recognize a large number of live cells plated in tissue culture flasks and to follow each of them through the incubation period of several days (14). In this way, both the killed and surviving fraction of cells is measured, eliminating experimental errors due to counting, pipetting, diluting and plating and only statistical uncertainties remain. These can be minimized by assaying for large numbers of cells employing binomial statistics. In previous studies at low doses, it was shown that the effect of radiosensitizers is greatly reduced below 3 Gy.

METHODS

AND

MATERIALS

The effect of truns-DDP on high and low dose radiation response was determined in a similar fashion to Skarsgard

* Medical Biophysics. t Cancer Imaging.

Reprint requests to: K. A. Skov, Ph.D. Accepted for publication 16 November 1281

1988.

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of truns-DDP

in hypoxic

May 1989, Volume

cells in comparison Enhancement

16, Number

with electron

5

affinic radiosensitizers

radio

Low dose (0.8% s)”

High dose (0.01% s)”

Trans-DDP 0.05 mM (+)” 0.05 mM (+) 0.075 mM (+) 0.10 mM (+)

1.2 f 0.1 2.2 + 0.2 1.5 1.6

1.1 + 0.1 1.5 + 0.1 1.2 1.3

2.0 2.4 2.5 2.0

Oxygen (this study)

2.4 f 0.2

3.1 + 0.2

0.7

2.4

3.5

Sensitizer (+) with (-) without pretreatment’

Oxygen ( 13)

Rel. effectiveness (low/high dose)‘”

(.65-.h74 (18)) Miso 1.5 mM (-) (12) 3.0 mM (+)”

1.3 1.8

1.9 2.3

.3 .63

SR-2508

1.7

2.2

.56

2.2

3.2

.53

3.0 mM (+) (18)

Ro-03-8799

0.5 mM (+) ( 18)

I 50-60 (18) minutes incubation with drug at 37” prior to irradiation. ‘I Enhancement ratio in hypoxic V-79 cells, calculated from survival after normalization for any measurable toxicity. i” Relative effectiveness = (ER,,., ~ I)/(E&,,, - 1) Skarsgard CI al. (18). ” Marginally toxic (survival 0.6-0.8). ” Significantly toxic (survival lower than 0.3). “’Skarsgard ef al., The effect of pretreatment on sensitization by misonidazole at low radiation doses, Abstract Di-1; presented Thirty-fifth Annual Meeting of the Radiation Research Society, Atlanta, Georgia, February 2 l-26, 1987. et al. (18). V-79 cells were grown in monolayers in Eagle’s MEM* with 10% FE%*. After trypsinization, the cells were placed under optimum growth conditions in suspension for 1 hr at 37°C. The cells were then suspended under hypoxia with or without drug and further incubated under flow of oxygen-free nitrogen for 1 hr at 37°C. Immediately thereafter, temperature was lowered to 4”C, and the cells were irradiated? to graded doses of X rays with the drug still present. Radiation response at high doses and toxicity of trans-DDPS were assessed using conventional clonogenie methods ( 10). For determining radiation response at low dose, the Cell Analyzer (14) was also used to follow individual cells from the time of platingg to the time of scoring. Survival (colony formation) or death (small colony or cell lysis) was determined after 6 days. The same cell suspensions were used for low and high radiation dose studies. In early experiments, trans-DDP at 0.05 mM exhibited variable toxicity. This was due to the differences in the pH of the medium during the 1 hr post-trypsinization incubation, even prior to drug exposure. If pH were strictly maintained at 7.1-7.2, the toxicity of trans-DDP was relatively low-plating efficiency of 0.6-0.8 (rel. to control) which we refer to as “marginally” toxic. If the pH of the medium rose to 7.4-7.5, the complex was more toxic with

* Gibco (Grand Island Biological Co.) of Canada, Burlington, Ontario. t X-ray source, Philips RT 250, 250 kV, 0.5 mm Cu, 5.37 Gy/min.

at

PE at 0.3-0.4, which we call “significantly” toxic. We have not attempted to study the pH/toxicity interaction at this time. We note, however, that a lack of effect of order-drug (cis-DDP) and trypsinization-was reported (8) and that there was not such a marked effect of pH on toxicity of cis-DDP during drug treatment (D. Wurst, K. Skov, unpublished data, June, 1983). In subsequent experiments, higher drug levels were used to achieve toxicity in cells which had been exposed only to pH 7.1 to 7.2.

RESULTS Figures 1 and 2 show the result of radiosensitizing activity of trans-DDP at high and low radiation doses. In Figure 1, a drug concentration was chosen which was significantly toxic to V-79 cells (survival of approximately 0.3). At that concentration, truns-DDP radiosensitizes the cells at high and low doses, although the enhancement ratio (ER) is much greater at low doses than at high doses. When cytotoxicity was marginal (survival -0.7), there is no significant radiosensitization noticeable at high doses, while at low doses it is still significant (Fig. 2). Table 1 summarizes the data of all experiments and also provides comparisons with other radiosensitizers previously studied. The relative effectiveness (18) clearly

$ Trans-DDP, 9 Plasticware,

Sigma Chem. Corp., Milwaukee, Falcon.

WI.

TrmwDDP and low radiation doses 0 K. A. SKOV et al.

1283

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DOSE

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18

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28

25

38



Fig. 1. Significantly toxic truns-DDP. V-79 cells were exposed to 0.075 mM truns-DDP for 1 hr at 37°C under hypoxic conditions and were then exposed to graded doses of 250 kVp X rays. Immediately after irradiation, the cells were made aerobic, washed free of the drug and were then plated. High dose data (right) were obtained using conventional survival assay while low dose assay (left) was that ofthe DMIPS Cell Analyzer. All data are normalized to S = 1.O at D = 0. Error bars represent standard error. Hypoxic cells without Cl or with v truns-DDP; dotted line represents oxic response.

that truns-DDP is a much better radiosensitizer at low than at high doses because the relative effectiveness is greater than two for all cases. Any other radiosensitizer, including oxygen, shows relative effectiveness less than one. That includes pre-incubation (i.e. cytotoxicity) effects of misonidazole, SR-2508, Ro-03-8799, and others. indicates

DISCUSSION Trans-DDP is the first radiosensitizer which exhibits greater radiosensitizing activity at low doses than at high

DOSE



radiation doses. Our current hypothesis is that this radiosensitizer affects only the shoulder region of the survival curve. This is consistent with studies using cisplatin which found that fractionated radiation regimens were more effective than single large exposures when the drug was employed (3). Studies on PLDR and SLDR using Pt complexes (2,3,7) also suggest that the effect is in the shoulder region of the cell survival curve. At present, we have not yet gathered sufficient experimental data to fully test this hypothesis, although the preliminary indications support it. The effect of this and other Pt compounds of clinical

DOSE

CGrav>

Fig. 2. Marginally toxic truns-DPP. Cells were treated the same as described in the caption to Figure 1, except that 0.05 mM truns-DDP was used in low dose (left) and high dose (right) experiments. Hypoxic cells without 0 or with v truns-DDP, dotted line represents oxic response.

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interest (cisplatin (3), carboplatin (6)) on parameters (Y and fl in a linear-quadratic fit of survival data at low and high doses is being examined. Metal complexes can sensitize by many mechanisms (19). At present, the mechanism by which platinum complexes interact with radiation in cells is not known (5) but, unlike nitroheterocyclic compounds, they do not interact via radiation free radical mechanism unless accompanied by a radiosensitizing ligand (1). To be active, Pt complexes have to bind to DNA and thus some pre-incubation with cells is used. It is possible that this type of drug inhibits repair mechanism(s) operating in the shoulder region. We plan to address this question at O-4 Gy using synchronized cells and especially, plateau phase cells. Alternatively, it may be simply a type of toxicity

May 1989. Volume which

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with

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that a preliminary

However, study

it is of considof the interaction

trans-DDP and oxic radiation showed no radiosensitization at low doses. When confirmed and extended to other Pt complexes,* this observation may provide important clues to the mechanisms of these interactions. It has been suggested that the best sensitizer may be a cytotoxic agent (16). Thus, perhaps the cis isomer-a more toxic agent-will be even more effective in this respect. Given the data to date (Table l), it appears that the platinum complexes may be very useful in the low radiation dose region, possibly much better than electron-affinic radiosensitizers. For example, even when toxicity was permitted, nitroimidazoles were still less effective at low doses than at the higher doses. between

REFERENCES 1. Butler, J.; Hoey, B. M.; and Swallow, A. J. The radiation chemistry of some platinum-containing radiosensitizers and related compounds. Radiat. Res. 102: l- 13; 1985. 2. Carde, P.; Lava], F. Effect of cis-dichlorodiammine platinum II and X rays on mammalian cell survival. Int. J. Radiat. Oncol. Biol. Phys. 7:929-933; 1981. 3. Dewit, L. Combined treatment of radiation and cis-diamminedichloro-platinum(I1): a review of experimental and clinical data. Int. J. Radiat. Oncol. Biol. Phys. 13:403-426; 1987. 4. Douple, E. B.; Richmond, R. C. Platinum complexes as radiosensitizers of hypoxic mammalian cells. Br. J. Cancer 37(Suppl. 111):98-102; 1978. 5. Douple, E. B.; Richmond, R. C. A review of platinum complex biochemistry suggests a rationale for combined platinum-radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 5: 13351339; 1979. 6. Douple, E. B.; Richmond, R. C.; O’Hara, J. A.; Coughlin, C. T. Carboplatin as a potentiator of radiation therapy. Cancer Treat. Rev. 12:ll l-124; 1985. 7. Dritschilo, A.; Piro, A. J.; Kelman, A. D. The effect of cisplatinum on the repair of radiation damage in plateau phase Chinese hamster (V-79) cells. Int. J. Radiat. Oncol. Biol. Phys. 5: 1345-l 349; 1979. 8. Durand, R. E. Chemosensitivity testing in V79 spheroids: drug delivery and cellular microenvironment. J. Nat. Cancer Inst. 77~247-252; 1986. 9. Hill, J.; Speer, R. J.; Loeb, E.; McLellan, R.; Hill, N. 0.; Kahn, A. Clinical experience with cisplatinous diamminedichloride (DDP). In: Advances in antimicrobial and antineoplastic chemotherapy, Vol. II. Baltimore: Univ. Park Press.; 1972: 125. 10. Moore, B. A.; Palcic, B.; Skarsgard, L. D. Radiosensitizing

* Korbelik, Skov and Palcic: presented at the 36th Annual Meeting Radiation Research Society, Philadelphia, PA, April 1988 (Abstract Fg-6); Accepted for publication in Radiation Research ( 1989).

1 I. 12.

13.

14.

15.

16.

17.

18.

19.

20.

and toxic effects of the 2-nitroimidazole Ro-07-0582 in hypoxic mammalian cells. Radiat. Res. 67:459-473; 1976. Nias, A. H. W. Radiation and platinum drug interactionreview. Int. J. Radiat. Biol. 48:297-314; 1985. Palcic, B.; Faddegon, B.; Skarsgard, L. D. The effect of misonidazole as a hypoxic radiosensitizer at low doses. Radiat. Res. 100:340-347; 1984. Palcic, B.; Jag& B. The use of solid state image sensor technology to detect and characterize live mammalian cells growing in tissue culture. Int. J. Radiat. Biol. 50:345-352; 1986. Palcic, B.; Jaggi, B. Image cytometry system for morphometric measurements of live cells. In: Wise, D. L., ed. Biosensors and bioelectronic systems. Boca Raton, FL: CRC: (In press). Palcic, B.; Skarsgard, L. D. Reduced oxygen enhancement ratio at low doses of ionizing radiation. Radiat. Res. 100: 328-339; 1984. Richmond, R. C.; Khokhar, A. R.; Teicher, B. A.; Douple, E. B. Toxic variability and radiation sensitization by Pt(I1) analogs in salmonella typhimurium cells. Radiat. Res. 99: 609-626; 1984. Rosenberg, B.; van Camp, L.; Krigas, T. Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature 205:698-699; 1965. Skarsgard, L. D.; Harrison, I.; Durand, R. E.; Palcic, B. Radiosensitization of hypoxic cells at low doses. Int. J. Radiat. Oncol. Biol. Phys. 12:1075-1078; 1986. Skov, K. A. Modification of radiation response by metal complexes: a review with emphasis on nonplatinum studies. Radiat. Res. 112:217-242; 1987. Zak, M.; Drobnik, J. Effects of cis-DDP on post irradiation lethality in mice after irradiation with x-rays. Strahlentherapie 142:112-l 15; 1971.